Recombinant Human TrkC

Recombinant Human TrkC is a valuable tool in research applications focused on neurobiology, cancer, and cell signaling because it enables precise investigation of TrkC-mediated pathways, ligand interactions, and functional assays in a controlled, reproducible manner.

Key reasons to use Recombinant Human TrkC in research include:

• Neurotrophin Signaling Studies: TrkC is a high-affinity receptor for neurotrophin-3 (NT-3) and is critical for studying neurotrophin signaling, neuronal development, and survival. Recombinant TrkC allows for in vitro assays to dissect these pathways without interference from other cellular components.
• Cancer Research: TrkC is implicated in tumor biology, acting as both an oncogene and a tumor suppressor depending on context. Recombinant TrkC can be used to study its role in tumor growth, metastasis, and response to targeted therapies, especially in cancers where TrkC is overexpressed (e.g., breast cancer).
• Enzyme Kinetics and Inhibitor Screening: Recombinant TrkC is suitable for biochemical assays, including kinase activity measurements and high-throughput screening of small molecule inhibitors or therapeutic antibodies targeting TrkC.
• Protein-Protein Interaction Studies: Recombinant TrkC enables binding assays to characterize interactions with neurotrophins (such as NT-3) or other signaling molecules, and to map domains responsible for ligand specificity or downstream signaling.
• Blocking and Functional Assays: It can be used as a blocking reagent to inhibit endogenous TrkC signaling in cell-based assays, or as a positive control in kinase assays to validate pathway activation.
• Cross-Species Activity: Human recombinant TrkC is highly conserved and exhibits cross-species activity, making it useful for comparative studies in different model organisms.

Additional considerations:

• Reproducibility: Recombinant proteins provide batch-to-batch consistency, which is essential for quantitative and comparative studies.
• Customization: Variants (e.g., Fc chimeras, fragments) allow for tailored applications such as immunoprecipitation, surface plasmon resonance, or cell surface binding studies.

In summary, using recombinant human TrkC enables detailed, reproducible, and mechanistic studies of TrkC function in neurobiology, oncology, and drug discovery.

Yes, recombinant Human TrkC can be used as a standard for quantification or calibration in ELISA assays, provided it is sufficiently pure and its concentration is accurately known.

For ELISA quantification, the standard should ideally be a purified recombinant protein that matches the analyte detected by your assay. Recombinant Human TrkC is commonly supplied in lyophilized form, often with BSA as a carrier to enhance stability and shelf-life, which is suitable for use as an ELISA standard. If your assay is sensitive to carrier proteins, a carrier-free version is also available.

Key considerations for using recombinant Human TrkC as an ELISA standard:

• Purity and Identity: Ensure the recombinant TrkC is highly purified and corresponds to the same isoform or epitope recognized by your ELISA antibodies.
• Concentration Accuracy: The protein must be accurately quantified, typically by absorbance at 280 nm, BCA assay, or HPLC, to prepare a reliable standard curve.
• Reconstitution: Follow the manufacturer’s instructions for reconstitution, usually in PBS with 0.1% BSA or another stabilizer, to maintain protein integrity.
• Standard Curve Preparation: Prepare serial dilutions covering the expected range of TrkC concentrations in your samples. Most ELISA standard curves range from low pg/mL to ng/mL concentrations, depending on assay sensitivity.
• Matrix Matching: If possible, dilute the standard in the same buffer or matrix as your samples to minimize matrix effects.

Protocol Example:

1. Reconstitute recombinant Human TrkC as instructed (e.g., at 100 μg/mL in PBS with 0.1% BSA).
2. Prepare serial dilutions to generate a standard curve (e.g., 0–10,000 pg/mL).
3. Run the standard curve alongside your samples in each ELISA plate for accurate quantification.

Limitations:

• The recombinant standard must be compatible with the antibodies used in your ELISA (i.e., same isoform and epitope).
• Carrier proteins (e.g., BSA) may interfere with some assays; use carrier-free protein if necessary.
• The standard curve should be freshly prepared for each assay to ensure accuracy.

In summary: Recombinant Human TrkC is suitable as an ELISA standard if it is pure, accurately quantified, and matches the assay’s target. Always verify compatibility with your specific ELISA kit and follow best practices for standard curve preparation.

Recombinant Human TrkC has been validated for several key applications in published research, including bioassays, surface plasmon resonance, immunoprecipitation, immunocytochemistry, blocking assays, and kinase assays.

Validated Applications in Published Research:

• Bioassay: Used to study TrkC-mediated signaling, neurotrophin interactions, and cell survival/apoptosis mechanisms in various cell types, including human neuroblastoma cells and chicken cells.
• Surface Plasmon Resonance (SPR): Applied to analyze protein-protein interactions, such as the association of sortilin with Trk receptors, which enhances neurotrophin signaling.
• Immunoprecipitation (PcIP): Utilized for identifying protein complexes and functional mimics of neurotrophic ligands, including studies involving pathogens like Trypanosoma cruzi.
• Immunocytochemistry/Immunofluorescence: Used to detect TrkC expression and activation in transfected cells and tissue samples, including labeling cell surface populations and monitoring receptor activation states.
• Blocking Assay: Validated for use in assays that assess the inhibition of TrkC activity or ligand binding.
• Kinase Assay: Used to measure TrkC tyrosine kinase activity, often in the context of neurotrophin signaling or drug screening.

Additional Context:

• Recombinant TrkC proteins have been employed as standards in ELISA and as reagents in cell/tissue culture experiments to probe neurotrophin receptor biology.
• TrkC agonist antibodies and recombinant proteins have been investigated for therapeutic potential in neurodegenerative and neuromuscular disease models, such as spinal muscular atrophy with respiratory distress (SMARD1).
• Immunofluorescence protocols have specifically used recombinant TrkC to label and quantify cell surface receptor populations in neuroblastoma research.

Summary Table of Applications

These applications demonstrate the versatility of recombinant human TrkC in both basic and translational neuroscience research.

Reconstitution Protocol

Reconstituting lyophilized recombinant human TrkC protein requires careful attention to preserve protein activity and functionality. Begin by centrifuging the vial at 5,000×g for 5 minutes before opening to concentrate the lyophilized powder at the bottom of the tube. This step is critical to prevent loss of material.

Add sterile distilled water or an appropriate buffer to achieve your desired working concentration. The recommended reconstitution concentration ranges from 0.1 to 1.0 mg/mL, depending on your experimental requirements. For example, if you have 100 µg of protein and want a final concentration of 100 µg/mL, add 1 mL of sterile water or buffer. Allow the vial to reconstitute for 15-30 minutes at room temperature with gentle agitation. Avoid vigorous vortexing or shaking, as this can cause foaming and protein denaturation.

Buffer and Carrier Protein Considerations

The specific buffer composition depends on your TrkC construct variant. For standard formulations, reconstitute in sterile PBS containing at least 0.1% human or bovine serum albumin (BSA) as a carrier protein. If your protein is formulated as carrier-free, reconstitute in sterile PBS without additional carrier proteins. For animal-free applications, 5% trehalose is recommended as an alternative carrier.

Consult the Certificate of Analysis (CoA) or product data sheet accompanying your specific protein preparation, as reconstitution requirements may vary based on the expression system, purification tag (His, Fc, or His-Fc fusion), and formulation.

Storage of Reconstituted Protein

Once reconstituted, store working aliquots at -20°C to -80°C for extended storage. Short-term storage at 4°C is permissible for 2-7 days. Critically, avoid repeated freeze-thaw cycles, as these significantly affect protein stability, pH, and can cause protein denaturation. Prepare small aliquots for individual experiments to minimize freeze-thaw exposure.

Pre-Experiment Preparation

Before use in cell culture experiments, dilute your reconstituted TrkC stock into the appropriate cell culture medium or experimental buffer to achieve the desired working concentration. For bioassays and functional studies, typical working concentrations range from 0.05-0.2 µg/mL in the presence of appropriate ligands such as neurotrophin-3 (NT-3). Ensure all solutions are sterile and handle the protein aseptically to maintain viability for cell culture applications.